Nucleic acid sequence data is valuable in myriad applications in biological research and molecular medicine, including determining the hereditary factors in disease, in developing new methods to detect disease and guide therapy (van de Vijver et al. (2002) “A gene-expression signature as a predictor of survival in breast cancer,” New England Journal of Medicine 347: 1999-2009), and in providing a rational basis for personalized medicine. Obtaining and verifying sequence data for use in such analyses has made it necessary for sequencing technologies to undergo advancements to expand throughput, lower reagent and labor costs and improve accuracy (See, e.g., Chan, et al. (2005) “Advances in Sequencing Technology” (Review) Mutation Research 573: 13-40, Levene et al. (2003) “Zero Mode Waveguides for Single Molecule Analysis at High Concentrations,” Science 299: 682-686).
Methods of preparing templates for large-scale sequencing projects have typically entailed constructing shotgun libraries that comprise overlapping fragments of, e.g., a genomic DNA; transforming cells with the library; growing cells to amplify each library member; and isolating and purifying library DNA. For example, shotgun cloning was initially used to prepare nucleic acid templates for sequencing small genomes such as that of the cauliflower mosaic virus (CMV) (Gardner, et al. (1981) “The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing.” NAR 9: 2871-2888). More recently, this template preparation strategy has been used to produce templates for the sequencing of complex genomes, including the mouse, Drosophila, and human genomes (Mural, et al. (2001) “A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome.” Science 296: 1661-1671; Adams, et al. (2000) “The genome sequence of Drosophila melanogaster.” Science 287: 2185-95; Venter, et al. (2001) “The Sequence of the Human Genome.” Science 291: 1304-1351).
However, the cloning and cell culture techniques used in shotgun library construction methods are time consuming, labor-intensive, costly, and not easily amenable to automation. Purification protocols that are used to isolate cloned nucleic acid templates from cells, e.g., bacterial cells, for sequencing do not reliably produce nucleic acid samples that are sufficiently free of sequencing reaction inhibitors such as salts, carbohydrates and/or proteins. Furthermore, these disadvantages are exacerbated when conventional template preparation methods are scaled to the quantities that would be useful for high throughput sequencing technologies, e.g., single-molecule real-time (SMRT) sequencing systems, such as those described in, e.g., Levene et al. (2003) “Zero Mode Waveguides for single Molecule Analysis at High Concentrations,” Science 299: 682-686; and Eid, et al. (2009) “Real-Time DNA Sequencing from Single Polymerase Molecules.” Science 323: 133-138.
Consequently, there is an increasing demand for efficient, low-cost methods for the preparation of high-quality nucleic acid templates for next generation sequencing technologies. The present invention provides methods and compositions that would be useful for supplying high throughput DNA sequencing systems with such templates.